PROJECT SUMMARY/ABSTRACT The cellular and molecular mechanisms underlying the telomerase-independent Alternative Lengthening of Telomeres (ALT) telomere maintenance mechanism is an area of active research, particularly in non-epithelial cancers where ALT is commonly found (e.g. gliomas, sarcomas, pancreatic neuroendocrine tumors). For many of these cancers the prognosis is poor and therapeutic options are limited. Encouraging results from a recent pre-clinical study show a pharmacological inhibitor of the DNA Damage Response (DDR) protein, ATR, exhibits selective toxicity against osteosarcoma and glioblastoma cancer cells that utilize the ALT pathway for telomere maintenance, instead of up-regulating the enzyme, telomerase. Less appreciated and under- investigated are adenocarcinomas that employ the ALT pathway. We hypothesize that ALT-positive adenocarcinomas share molecular features with ALT-positive cancers of non-epithelial origin, and that these similarities will result in a comparable sensitivity to ATR inhibition. The overarching goal of this proposal is to study ALT in adenocarcinomas, using prostate cancer cell lines as an initial in vitro model system, to investigate whether similar strategies of pharmacologically inhibiting DDR proteins, such as ATR, in ALT-positive adenocarcinomas will also show efficacy. In Aim 1, we will generate and validate the first adenocarcinoma cell lines that employ ALT. Our laboratory previously discovered a strong correlation between ALT-positive tumors and somatic inactivating mutations in either the ATRX and DAXX genes. To date, no prostate cancer cell line has been identified that displays the ALT phenotype. Using the CRISPR cas9 genome editing technique, we will create isogenic prostate cancer cell lines with inactivating mutations in ATRX or DAXX genes. In Aim 2, we will characterize the molecular features underlying the ALT process in prostate cancer cells by comparing the telomerase-positive/ALT-negative parental lines for differences in the relative expression of telomerase components, telomerase activity, nuclear architecture, and activity levels of homology-directed recombination, which is thought to be crucial to the ALT pathway. Finally, in Aim 3, we will evaluate the response of ALT-positive prostate cancer cells to DDR inhibition. Preliminary evidence shows a dramatically activated DDR in ALT-positive cells, suggesting that an increase in the level of DNA damage or a reduction in repair capacity in these already stressed cells may be sufficient to induce apoptosis. DDR proteins will be inhibited using RNA interference strategies, as well as existing pharmacological inhibitors in ALT-positive prostate cancer cell lines. The research findings of the proposed study will have real biological and therapeutic value by increasing our understanding of the molecular mechanisms underlying ALT in adenocarcinomas, and by determining if this unique molecular subset of carcinomas is susceptible to enhanced killing by inhibiting DDR proteins.